Cell, the
basic unit of life depends for its survival on nutrients and thereby energy to
perform its physiological function. Cells of lymphoid and myeloid origin are
key in evoking an immune response against “self” or “non-self” antigens. The
thymus derived lymphoid cells called T cells are a heterogenous group with
distinct phenotypic and molecular signatures that have been shown to respond
against an infection (bacterial, viral, protozoan) or cancer. Recent studies
have unearthed the key differences in energy metabolism between the various T
cell subsets, natural killer cells, dendritic cells, macrophages and myeloid
derived suppressor cells. While a number of groups are dwelling into the
nuances of the metabolism and its role in immune response at various strata,
this review focuses on dynamic state of metabolism that is operational within
various cellular compartments that interact to mount an effective immune
response to alleviate disease state.

INTRODUCTION

Cells depend on
nutrients available in their extracellular environment to support the
biochemical processes that are required for cell growth and proliferation. The
cells responsible for mounting adaptive immunity in response to pathogens or
cancers require a set of complex but coordinated signals to drive their
activation, proliferation, and differentiation. It is increasingly clear that
all cell types have cellular metabolism coupled with various stages in their
life-span to meet the energetic requirements for survival. A comprehensive
understanding about the role of metabolism in cellular function is therefore
important for developing novel therapeutic approaches to treat various diseases
or cancer. Here, we discuss briefly recent studies that highlight the role of
metabolic pathways or metabolites in the function of both lymphoid and myeloid
cells.

Immunometabolism
of Lymphoid Cells

T cell: The activation of the naïve T cell either through T
cell receptor (TCR) engagement (or) by a mitogen leads to numerous changes in
its proliferation/expansion and renders the activated T cells with distinct
phenotype and function [1]. T cell activation also leads to rapid shifts in
cell metabolism to co-opt the bioenergetic needs of a rapidly proliferating T
cell [2]. Quiescent T cells are in continuous need for cellular energy provided
by adenosine triphosphate (ATP) consumption for their migration and persistent
cytoskeletal rearrangement; therefore they rely preferentially on the
growth-promoting pathways as oxidation of pyruvate, fatty acid and glutamine
[2]. Early study by Rathmell et al. showed that in the absence
of extrinsic signals, nutrient utilization by lymphocytes is insufficient to
maintain either cell size or viability [3]. Their study demonstrated that after
TCR engagement was lost, lymphocytes rapidly down regulated the glucose
transporter, Glut1 along with reduced mitochondrial potential and cellular ATP.
Another study from Craig Thompson’s group showed that second signal in form of
co-stimulation leads to bioenergetics modulation that results in a decision on
anergic vs. effector T cell response [4]. Further, work by
Jonathan Powell’s group elegantly showed that anergic T cells are in fact
metabolically anergic as well [5]. An important observation from Thomas
Gajewski’s group showed that effector cytokine secretion by activated T cells
is dependent on availability of glucose, and inhibiting glycolytic pathway
using 2-deoxyglucose (2-DG) results in loosing cytokine secretion [6].

Thus, these
pioneering studies firmly established that glucose metabolism in lymphocytes is
a regulated process that effects on immune cell function and survival [7].
Activation of T cells not only results in increase in Glut1 expression and
surface localization, but if glucose uptake is limited, glycolytic flux
decreases to a level that no longer sustains viability, and proapoptotic Bcl2
family members become activated, promoting cell death [7].

T cell subsets
and metabolism:

Given the
heterogenous phenotype of both CD4+ T helper (Th) and CD8+ T
cytotoxic (Tc) cells that also differentiate to distinct lineages based on
effector cytokine secreting signature (i.e.Th1/Tc, Th2/Tc2, Th9/Tc9,
Th17/Tc17, Treg’s), it is important to determine if all these T cell subsets
follow similar or unique metabolic signature. Seminal studies from Rathmell’s
group showed that Th1,Th2, and Th17 cells strongly engage glycolysis, whereas
tolerance inducing regulatory T cells (Treg’s) depend more on the oxidative
phosphorylation to fulfill their bioenergetics demands [8]. Similar to CD4+ T
cell, CD8+ T cells that differentiate to effector cytolytic T
cells following activation preferentially use glycolysis as their major
bioenergetic pathway [9], whereas, the small percentage of CD8 T cells which
persist as memory cells after contraction of the effector phase mostly rely on
the oxidative phosphorylation for energy [10]. Primarily, glycolysis is an
anaerobic metabolic pathway happening in the cytosol. It degrades glucose to
yield pyruvate and other precursors needed for cellular anabolism. For every
molecule of glucose metabolized through the glycolytic pathway two molecules
ATP are produced. Activated effector T cells convert most of the pyruvate, a
downstream product of glycolysis, into lactate instead of Acetyl-CoA that can
be oxidized in mitochondria [2]. Even though glycolysis has low yield of ATP,
it is considered to be a preferable pathway over oxidative phosphorylation,
which has high ATP output, for activated effector T cells. This switch into
glycolysis by stimulated T cells is important for NADPH production and
nucleotide synthesis which are needed by proliferating T cells. Recently, a
glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been
shown as a post-transcriptional inhibitor of interferon gamma mRNA and once
GAPDH engages in glycolysis it releases IFNγ mRNA leading to IFNγ production
[11]. Even though glycolysis pathway is important for activated effector T
cells, mitochondrial pathway is not completely inhibited. Instead, glutamine
oxidation by mitochondria is enhanced in activated T cells to replenish
tricarboxylic acid cycle (TCA) and produce reactive oxygen species (ROS), which
has been proven to be important for activation of T cells and signaling of
interleukin 2 production [12]. Oxidative phosphorylation (OXPHOS) is the major
pathway of producing ATP. Glucose, glutamine and fatty acid are oxidized in
mitochondria via TCA cycle to generate reducing equivalents, such as
nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide
(FADH2). These molecules then feed the OXPHOS pathway by donating electron to
electron transport chain (ETC). OXPHOS pathway are favored by regulatory T
cells (Treg’s) and memory T cells for their development and long-term survival [13].
Also, memory T cells maintain greater mitochondrial mass than effector T cells
which indicate their dependence on OXPHOS pathway [14]. Enhancement of fatty
acid oxidation promotes the development of memory CD8+ T cells after
immunization [15-19]. Thus, the changes in metabolism between glycolysis and
OXPHOS pathway are mandatory to maintain T cell function and efficient
progression from naïve phenotype to effector and memory phenotype.

It is also
important to understand the underlying mechanisms of adaptation of distinct
metabolic programming in effector vs. Treg (or memory T)
cells following encountering immunological signals which drive them into
different functional subsets. Recent studies have shown that effector T cells
express high surface levels of the glucose transporter Glut1 that makes them
highly glycolytic [9]. In contrast, Treg’s express low levels of Glut1 and have
high lipid oxidation rates [8]. It has been shown that blocking glycolysis
inhibits Th17 development while promoting Treg cell generation [20]. Further,
it has been also shown that the effector T cells exhibit the metabolic
phenotype that is not fixed [21]. However, the state is changeable or dynamic
between the OXPHOS and Glycolysis. Upon activation, mitogen-activated T cells have
been documented to switch to glycolysis, less sufficient pathway of energy
production, to support their biosynthesis processes [8]. Some of the activated
T cells survive to form long lived memory T cells and switch to β-oxidation of
fatty acid [22]. Similarly, regulatory T cells have shown high lipid oxidation
in vitro [8]. The fate of an activated T cells depend on many factors such as
the strength of TCR signaling, costimulatory molecules and cellular
microenvironment. Cellular microenvironment is represented by nutrition and
oxygen level surrounding activated T cells. These factors highly affect
mammalian target of rapamycin signaling pathway (mTOR). Suboptimal signaling of
mTOR pathway during starvation results in generation of Tregs and inhibition of
effector T cells [23]. Rapamycin, inhibitor of mTOR pathway, has similar
effect, it inhibits T cell proliferation and selectively increase Treg
generation [24]. Glut, glucose uptake receptor, transgenic T cells have shown
more proliferation and cytokine production; Treg’s, in contrast, had normal
response [8]. Fatty acid oxidation has equal importance for Treg’s and memory T
cells as glucose uptake for activated effector T cells. It has been found that
inhibition of lipid oxidation through CPT1a, carnitine palmitoyltransferase 1A,
compromises Treg development, whereas increase provision of lipid either
suppresses effector T cells or enhances Treg’s proliferation [8]. Unlike Treg’s
which use extracellular lipid to produce phospholipid used in the formation of
cellular membrane, Th17 cells use endogenous fatty acid synthesis for the same
purpose [25]. Each T cell subset has its own metabolic signature that can be
targeted by developing a pharmacological drug that can help either in
enhancement or suppression of T cell function. Therefore, it is likely that
transition from one metabolism pathway to another dictate T cell functions and
shapes their different subsets. This phenomenon has been employed by tumor
cells through high consumption of glucose, an important substrate for effector
T cells, in their microenvironment. Also, tumor cells bind programmed death
molecule-1 (PD-1) on exhausted T cells which target downstream signaling
leading to a decrease in glucose metabolism.

With regards to
the role of metabolism in pathogenic T cells, it has been shown that
alloreactivity T cells in graft versus host disease (GVHD) enhance both
glycolysis and OXPHOS pathway with low glutathione and high reactive oxygen
species (ROS) production [26]. Increasing ROS production by using Bz-42, an
inhibitor of mitochondrial F1F0-ATPase, selectively induced apoptosis in
alloreactivity T cells but not resting T cells or proliferating bone marrow
cells [27]. Rapamycin has long been used as a potent immunosuppressive therapy
in transplantation by targeting mTOR pathway [28], a central regulator of T
cell activation. TCR-dependent signaling of mTOR pathway leads to Glut1
increase, and subsequently glucose uptake associated with effector T cell
phenotype. Also, mTOR downstream signaling of HIF1α which further induces
glycolytic pathway selectively enhances Th17 development over Treg by
activating transcription factor RORγ [20]. Likewise, AMP-activated protein
kinase (AMPK) activation, which enhances fatty acid oxidation (FAO) also alters
this balance in favor of Treg cells [29]. HIF1α deficient mice have diminished
Th17 development but enhanced Treg cell differentiation and protected mice from
autoimmune neuroinflammation [20]. Furthermore, inhibition of acetyl-CoA
Carboxylase 1(ACC1) used by Th17 for endogenous fatty acid synthesis attenuates
Th17 mediated autoimmune disease.

The inhibition
of pyruvate dehydrogenase kinase (PDK1) enzyme, which is upregulated during T
cell activation to prevent pyruvate from being oxidized in mitochondria and
rather forming lactate, has led to inhibition of collagen-induced arthritis in
female mice [30]. Moreover, PDK1 inhibition has an effect on human and mouse
asthma model by inhibiting lactate production, proliferation of T cells and
production of IL17 and IFNg on other hand, it stimulates the production
of IL -10 and the induction of Foxp3 [31]. During switching from effector T
cells toward memory T cells, memory CD8 T cells switch to fatty acid oxidation
and down regulate glycolysis. High glycolysis uptake in CD8 T cells has been
shown to be associated with compromise in the generation of long-lived memory
cells by driving T cells toward a terminally differentiated state [32]. In the
same study, it has been reported that Pmel (pre-melanosome protein) transgenic
CD8 T cells co-cultured with 2DG, glycolysis pathway inhibitor, have better
antitumor effect in adoptive T cell therapy, indicated by smaller tumor size
and longer survival time [32]. To obtain a robust long lived CD8 memory T cells
many studies showed that enhancement of fatty acid oxidation by treating CD8 T
cells with either metformin, activator of AMPK pathway, or inhibitor of mTOR
pathway has led to the development of CD8 memory T cell after immunization
[15-19]. Thus, regulating T cell metabolism is a promising target for
immunotherapy to maintain the balance of Treg and Teff cells and enhance CD8
memory T cells that are important in tumor adoptive T cell therapy.

Further, the
cytokines that help expanding T cells have been shown to play a role in
modulating T cell metabolism. Using the cytokines interleukin (IL)15 and IL-2,
it was shown by Pearce et al that memory T cells generated with IL15 exhibit
enhances spare respiratory capacity, as compared to the effector T cells
generated with IL2 [33]. This study showed that IL15, a cytokine critical for
CD8+ memory T cells, regulated spare respiratory capacity and oxidative
metabolism by promoting mitochondrial biogenesis and expression of carnitine
palmitoyl transferase (CPT1a), a metabolic enzyme that controls the
rate-limiting step of mitochondrial fatty acid oxidation (FAO). These results
established how cytokines control the bioenergetic stability of memory T cells
by regulating mitochondrial metabolism. It has also been recently shown that
IL7, that plays an important role in homoeostatic proliferation and
differentiation, induces expression of the glycerol channel aquaporin 9 (AQP9)
in virus-specific memory CD8+ T cells, but not naive cells, and that AQP9 is
vitally required for their long-term survival [33a]. AQP9 deficiency impairs
glycerol import into memory CD8+ T cells for fatty acid esterification and
triglyceride (TAG) synthesis and storage. These defects can be rescued by
ectopic expression of TAG synthases, which restores lipid stores and memory T
cell survival. This study uncovers the metabolic mechanisms by which IL-7
tailors the metabolism of memory T cells to promote their longevity and fast
response to rechallenge. Therefore, strategies to modulate metabolic pathway of
T cell subsets could result in growth enhancement/deterioration and
hyper-functionality or dysfunctionality with implications in autoimmune
diseases and cancer.

B cells: B cells originate in the bone marrow from
hematopoietic stem cells which give rise into multipotent progenitor (MPP)
cells, then common lymphoid progenitor (CLP) cells. CLP differentiates toward
natural killer cells, T cells and B cells. Unlike NK cells and T cells which
mediate cellular immunity, B cell main function is mediating humoral immunity.
B cells migrate from bone marrow to the spleen as immature B cells [34]. Once
they are in the spleen, they differentiate toward follicular B cells or
marginal zone B cells. There are many types of B cells, most common one is
follicular B cell (FB cell) that if not circulating through the body, reside in
the follicles of secondary lymphoid organ [35]. Following FB cell is marginal B
cell that resides in the marginal zone of lymph node and form the first defense
of pathogen encountered in the secondary lymphoid organ. After activation, B
cells mature toward antibody secreting plasmablast which later form long lived
plasma cells residing in the bone marrow. Recently, a new class of B cell is
discovered called memory B cell that is rapidly reactivated to produce antibodies.
While the understanding of B cell metabolism is not as abundant as T cells,
here we cover the recent studies that have demonstrated the metabolic
regulation of B cell responses.

The metabolic
re-programming of activated immune cells now is gaining more attention and
dramatically regulates immune cell function. As in T cells, AKT/PI3K pathway
plays a key role in upregulation of Glut1 receptor to enhance glucose uptake
upon antigen stimulation in B cells and blocking this pathway prevents BCR
mediated growth [36]. Glycolysis plays a key role in T cell proliferation,
activation and cytokine production [37]. Similarly, B cell engaged glycolysis
and formed lactate when it was measured at 24hrs and 48hrs [36], showing a
preference for glycolysis pathway over OXPHOS. The FcγRIIB is a potent
inhibitory co-receptor that blocks BCR signaling in response to immune
complexes and, as such, plays a decisive role in regulating Ab responses [38].
It is noteworthy that co-ligation of the BCR and FcγRIIB has led to almost 82%
reduction in glucose uptake when compared with B cells stimulated via the BCR
alone [36]. This shows that glycolysis is a key player in B cell activation.
While, activated T cells and tumor cells show predominant transition into
glycolysis with low OCR/ECAR ratio [33, 39-41], the OCR/ECAR ratio was
found to remain unchanged in B cells after stimulation with LPS [42].
Consequently, B cells are unique and increase both glycolytic and mitochondrial
metabolic activity in a balanced fashion after LPS stimulation [43]. Unlike
Th17 cells, B cells do not require HIF1α to induce glycolysis- as B cells from
HIF1α deficient mouse induced glycolysis as comparable to wild type mouse after
LPS activation [20,43]. In contrast to HIF1α, c-Myc, a transcription factor
important for cell proliferation, has been shown to enhance glycolysis gene
expression and glutamine metabolism in both activated T cell and B cell
[43,44]. It was demonstrated that Myc-deficient B cell failed to increase ECAR
rate as well as OCR [43]. In the same study, lipid oxidation decreased whereas
pyruvate oxidation increased after LPS activation [43]. Compromising aerobic
glycolysis pathway by either treating B cell with glucose inhibitor, pyruvate
dehydrogenase kinase inhibitor or down-regulating Glut1 receptor leads to
suppression of Ab production. This indicates that Ab production by B cells is
controlled by glucose substrate as that of IFNγ by T cells.

Cytokines play
a role in the activation and programming of immune cells. B cell activating
factor (BAFF), secreted by myeloid cells, maintains B cell survival and
differentiation [45]. It acts as a co-stimulatory signal in B cell activation.
Chronic exposure to BAFF leads to increase metabolic capacity of B cells.
Glucose uptake as well as ECAR rate increased after 6hrs activation with LPS in
BAFF transgenic B cells [43]. BAFF signals B cell through PI3K/AKT pathway
[46], and consequently leads to increase glucose uptake. Expose B cells to
elevated levels of BAFF leads to a spontaneous SLE-like disease in mice [47].
As a result, BAFF seems a promising target in autoimmune disease.

In the
intestine, naive B cell residing in the Payer’s patch differentiate into IgA
producing plasma cells that migrate to intestinal Lamina propria (iLP).
Kunisawa et al. has shown that naïve B cells depend on TCA cycle and Vitamin B,
enzymatic cofactor for TCA cycle’s enzymes, for their energy production [48].
Inhibition of TCA cycle or deletion of Vitamin B1 from the
nutrient result in a low number of naïve B cells as well as prevent their
proliferation [49]. On the other hand, IgA plasma cells (PCs) in the iLP obtain
energy (ATP) from both glycolysis and TCA cycle. PCs are not intensely affected
by inhibiting OXPHOS pathway or deletion of vitamin B1. This study
shows that different B cell subsets have a distinct metabolic pathway, which is
poorly understood and needs further investigation.

Natural Killer
(NK) Cell: NK Cells have been known to
be the first line of defense against the invading non-self or self-antigens
[50]. Recent studies have now started to dissect the role of metabolism in NK
cell mediated effector and memory responses [51], which we briefly discuss
here. It has been shown that mTORC1-dependent metabolic reprogramming is a
prerequisite for NK cell effector function [52]. This study showed that NK
cells undergo dramatic metabolic reprogramming upon activation, up-regulating
rates of glucose uptake and glycolysis, and that mTORC1 activity is essential
for attaining this elevated glycolytic state. Directly limiting the rate of
glycolysis is sufficient to inhibit IFNγ production and granzyme B expression.
Similarly, it is shown that the metabolic checkpoint kinase mTOR was activated
and boost bioenergetic metabolism after exposure of NK cells to high
concentrations of IL15, whereas low doses of IL15 triggered only
phosphorylation of the transcription factor STAT5 [53]. However, another study
has shown that the degree of activation of NK cells regulate it’s metabolic
commitment [54]. This study investigated the metabolic requirements
for production of IFNγ by freshly isolated NK
cells, and showed significant differences in the metabolic requirements of
murine NK cell IFNγ production depending upon the activation signal [54]. A
striking result was that stimulation of NK cell IFNγ production was independent
of glycolysis or mitochondrial oxidative phosphorylation when cells were
activated with IL12 plus IL18. However, stimulation via activating NK receptors
required glucose-driven oxidative phosphorylation. Importantly, prolonged
treatment with high-dose, but not low-dose, IL15 eliminated the metabolic
requirement for receptor stimulation. Thus, these handful studies on NK cell
metabolism indicate that with a complex network of activation and inhibitory
receptors there may be an important role of metabolic regulators that control a
robust NK cell activation, exhaustion or memory response.

Immunometabolism
in Myeloid Cells

In addition to
lymphoid cells, a good bit of recent literature highlights the importance of
understanding metabolism in the myeloid cells as dendritic cells, macrophages
or myeloid derived suppressor cells. We discuss here briefly about the role of
metabolism in shaping the phenotype and function of myeloid cells.

Dendritic Cell: Dendritic cells (DCs) have been known as the
professional antigen presenting cells that respond to pathogens or other danger
signals and initiate innate and adaptive immune responses. An increased
understanding of DC metabolism under different disease states such as infection
or tumor is important to understand how the innate effectors get activated or programmed to respond and control such pathological conditions.
A recent study from the Edward Pearce’s group showed that stimulation of TLR
induces a metabolic transition in resting immature DC, characterized by a
conversion from mitochondrial β-oxidation of lipid and OXPHOS to aerobic
glycolysis [55]. It was a surprising finding that DCs that do not undergo
robust proliferation (as compared to tumor cells or T cells) also depend upon
glucose availability for optimal maturation (as seen by upregulation of CD40,
CD80, and CD86) and survival. It is thus likely that DCs in tumor
microenvironment are rendered dysfunctional in terms of antigen presentation or
secreting immunogenic cytokines as they compete for glucose substrate with the
highly glycolytic tumor cells. Similar to cancer cells and effector T cells,
PI3K/AKT pathway has been shown to play a key role in controlling metabolic
transition to glycolysis in TLR-stimulated DC [55]. AKT promotes glycolysis in
DC in part by increasing the expression of Glut-1 and likely activates
downstream mTOR pathway. However, some reports have shown that inhibition of
mTOR by rapamycin in murine GM-CSF-driven DC and human myeloid DC prolong the
lifespan, promote expression of co-stimulatory molecules and cytokines, and
enhances DC immunogenicity [56]. It is likely that the persistent stimulation
signals in form of pro-inflammatory cytokines or duration of Toll like-receptor
(TLR) engagement may have a role in level of mTORC1 or mTORC2 involvement
leading to differences in DC metabolic state and may account for variable
observations.

DCs that are
generated from a common myeloid progenitor in the bone marrow can also be
differentiated toward different DC subsets. To date, there are four identified
subsets of DCs: classical DCs, monocyte derived DCs, plasmacytoid dendritic
cells (pDCs) and Langerhans cells [57]. They are well-known as mediators of
innate and adaptive immune response rendering them critical for immunotherapy.
As a result, large focus has been recently shed on the metabolism of DCs
subtypes. In vitro differentiation of monocyte derived DCs and in vivo
development of DCs require fatty acid synthesis [58]. On the contrary, fatty
acid synthesis blockade enhanced bone marrow derived DCs immunogenicity by
showing increase antigen uptake, pro-inflammatory
cytokine production, and priming of Ag-restricted CD4+ and CD8+ T cells [58].
These results indicate that DCs function is tethered to their metabolism that
can be a promising target for immunotherapy. Peroxisome proliferator receptor-γ
(PPARγ), mediator of fatty acid metabolism, along with mitochondrial biogenesis
regulator, PPARγ co-activator 1 α is shown to be increased in in vitro
differentiated monocyte-derived DCs [59,60]. Also, inhibiting electron
transport chain (ETC) has led to a prevention of DCs differentiation from
monocyte which can be reflected by the low expression of CD1a differentiation
marker [61]. The development of cDCs and pDCs from committed dendritic cells
progenitor (CDP) depends on cytokine signaling from FMS-like tyrosine kinase 3
(Flt3) receptor expressed by CDP [62]. Also, providing CD8+ DCs and their
corresponding CD103+ tissue DCs with Flt3 cytokine ligand (Flt3L) leads to
increase in their expansion [63]. Rapamycin mediated inhibitor of mTORC1 compromises
Flt3 ligand signaling leading to prevention of pDCs and cDCs growth in vitro as
well as in vivo [64, 65]. Moreover, phosphate and tensin homologue (PTEN), a
negative regulator of mTOR pathway, deletion enhanced in vivo expansion of cDCs
and pDCS, an effect that can be abrogated by rapamycin [65]. MYC, a
transcription factor, is a downstream signaling of mTOR pathway and responsible
for gene expression of glycolytic protein [66]. It has been shown that Myc
paralogue, L-Myc, deficiency reduced the number of migratory CD103+ DCs,
beside splenic CD8+ and CD8- DCs [67]. In addition to the reduction of
number also antigen priming was lost in CD8+ DCs and pDCs [67]. One of the
genes that are targeted by L-Myc is NADH dehydrogenase (complex I) that impacts
the energy metabolism of DCs [67]. Resting bone marrow derived dendritic cells
(BMDCs) use both fatty acid oxidation in mitochondria and glycolysis when they
are induced by granulocyte macrophage colony stimulating factor (GM-CSF) [55].
However, it is not quite clear if cDCs and pDCs oxidize fatty acid as well. It
is now clear that monocyte derived DCs and cDCs increase glucose flux at early
stage of activation [55]. Hypoxia faced in inflammatory state along with Toll
like-receptor (TLR) activation has been shown to drive DCs shifting metabolism
toward glycolysis via activation of HIF1α [68]. Thus, dendritic cell metabolism
seems to undergo dynamic change to accommodate their microenvironment and
execute their function. Using 2-DG, inhibitor of glycolysis pathway, render DCs
inactive [69]. AMP-activated protein kinase (AMPK), which mediate inhibition of
mTOR pathway and enhance OXPHOS pathway [70,71], suppresses TLR-induced glucose
consumption and consequently activation of DCs [72]. Even though, it seems that
DCs follow Warburg effect by increasing glucose uptake after activation, it has
been revealed that rapid incorporation of glucose-derived carbon into TCA cycle
enhanced at early stage of activation [69]. The reason of glucose oxidation is
assumed to facilitate a transient increase in spare respiratory capacity [73]
and the use of TCA intermediate, citrate, for fatty acid synthesis [69]. After
12 hour of BMDCs activation, their metabolism almost entirely switches toward
glycolysis [55,74]. This is attributed to the production of nitrogen oxide (NO)
from inflammatory DCs which can block ETC. This might be one of the reasons
behind switching toward Warburg metabolism in the absence of increase
proliferation [74]. Interestingly, mTOR inhibition associated with iNOS down
regulation leads to increase DCs lifespan and maintain their mitochondrial
function [75,76]. The role of fatty acid synthesis in activated DCs is not
clear since these cells do not proliferate after activation. Nonetheless, the
necessity to increase the mass of Golgi and endoplasmic reticulum (ER) as a
result of increase protein synthesis could explain the demand for fatty acid
synthesis [69]. De novo fatty acid synthesis and lipid content
is correlated with immunogenicity of liver DCs [77]. On the contrary, high
lipid content decrease their immune priming function in tumor microenvironment
[78].

Eventually,
many factors affect the metabolism of DCs such as oxygen and nutrients
availability and microenvironment where DCs reside in, i.e. tumor or sites of
inflammation. However, it is poorly understood why DCs adopt fatty acid
synthesis at resting state and whether different DCs subsets have distinct
metabolic requirements.

Macrophages and
Myeloid-derived suppressor cells: Myeloid-derived suppressor cells (MDSC) are one of the major components
of the immunosuppressive network responsible for immune cell tolerance in
cancer [79-84]. Polarized MDSC lineages can be distinguished as M1 and M2
cells. M2 can be induced by interleukin (IL)-4 or IL-1, and produce arginase 1
and anti-inflammatory cytokines, eventually converging to facilitate
tumorgenesis [83-87]. In marked contrast, M1 could be induced by
lipopolysaccharide (LPS) or/and IFN-γ and produce inducible nitric oxide
synthase (iNOS), nitric oxide (NO), and pro-inflammatory cytokines, leading to
their antitumor effects [79, 80,85]. A recent study has determined the
mechanisms that underlie differentiation of MDSCs into M1 or M2 myeloid lineage
and their effect on cancer pathophysiology. They observed that glycolytic
activation through the SIRT1-mTOR/HIF -1α pathway was required for
differentiation to the M1 phenotype [88]. This implies that SIRT1 is a key
factor in the regulation of MDSC differentiation into M1 and M2 phenotypes
through hypoxia-inducible factor-1α (HIF -1α) –induced glycolytic metabolic
reprogramming and has an impact on MDSC functions in both immune suppression
and promotion of tumor progression. It was also established by another study
that functional polarization of tumor-associated macrophages is mediated by
tumor-derived lactic acid that is regulated by HIF1α [89]. In addition, the
lactate-induced expression of arginase-1 by macrophages was shown to play an
important role in tumor growth. Recently, another study showed that tumor-infiltrating
MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation
(FAO) [90]. This was accompanied by an increased mitochondrial mass, up
regulation of key FAO enzymes, and increased oxygen consumption rate.
Pharmacologic inhibition of FAO blocked immune inhibitory pathways and
functions in T-MDSC and decreased their production of inhibitory cytokines. FAO
inhibition alone significantly delayed tumor growth in a T-cell-dependent
manner and enhanced the antitumor effect of adoptive T-cell therapy.

Metabolic
Interactions, Redundancy and Immune outcome

The redundancy
or overlap of pathways involved in generating energy for the cellular functions
still exists. One of the previous study also shows that mitochondrial ATP is
essential for the rapid induction of glycolysis in response to activation and
the initiation of proliferation of both naïve and memory T cells [10]. While
this study reconfirmed the finding that similar to CD4+T cells, CD8+ memory T
cells also depend on fatty acid oxidation for bioenergetics requirements, they
also demonstrate that dissociation of the glycolysis enzyme hexokinase (HK)
from mitochondria impairs proliferation and blocks the rapid induction of
glycolysis upon T-cell receptor stimulation in memory T cells. It must be noted
that hexokinase-mitochondrial interaction has been shown to regulate glucose
metabolism differentially in adult and neonatal cardiac myocytes [91]. In this
adult myocyte model it was shown that while over expression of HKI, but not
HKII, increased glycolytic activity – demonstrating that differential
interactions of HKI and HKII with mitochondria underlie the different metabolic
profiles. Further, the role of Hexokinase-Mitochondria Interaction has been
established in Akt mediated inhibition of apoptosis [92]. This study showed
that targeted disruption of mitochondria-hexokinase (HK) interaction or
exposure to pro--apoptotic stimuli that promote rapid dissociation of
hexokinase from mitochondria potently induce cytochrome c release and apoptosis,
even in the absence of Bax and Bak. It is intriguing that despite the widely
appreciated anti-apoptotic activity of Akt that is coupled, at least in part,
to its effects on cellular metabolism-a study also showed that pharmacologic
inhibition of Akt enables expansion of potent tumor-specific lymphocytes with
the transcriptional, metabolic, and functional properties characteristic of
memory T cells [93]. While glycolytic pathway enzymes (as HKII or GAPDH) or
metabolites may be important for effector or memory T cell energetics, it is to
remember that end product of lactic acid that exits from the cell in the
microenvironment causes an unfavorable environment for normal cells. An earlier
study has shown that acidic pH results in T cell dysfunctionality and death
[94]. Overall, it is acceptable fact that the effector cytokine function of the
lymphoid cells is primarily dependent upon the glycolytic pathway. The
translation of key effector cell cytokine IFNγ has been shown to be regulated
by the sustained glycolysis, through glyceraldehyde 3-phosphae dehydrogenase
(GAPDH) with the 3’ untranslated region (UTR) of the IFNγ mRNA [95]. In
addition, inhibition of glycolysis or the use of alternative oxidative fuel as
galactose resulted in increased expression of immune inhibitiory receptor PD1,
which has been extensively shown to inhibit T cell response [95]. However,
since oxidative phosphorylation and spare respiratory capacity (SRC) has been
shown to be important for generation of T cell memory response [40], it remains
to be established if galactose cultured T cells that have higher oxidative
metabolism and SRC will be able to treat tumors.

Targeting
Metabolism

Dampening the
immune response using metabolic targets has already been tried in autoimmune
diseases. It has been shown recently by Yin et al. that the
two key metabolic pathways—glycolysis and mitochondrial oxidative
metabolism—are elevated in cells from SLE patients as well as in mouse models
of disease [96]. Using inhibitors of these pathways currently in the
clinic—2-deoxy-D-glucose (2DG) and metformin—normalized T cell metabolism and
decreased markers of SLE in animal models as well as in cells from SLE
patients. These data suggest that inhibiting both glycolysis and mitochondrial
metabolism could be a new therapeutic strategy for treating SLE. Similarly, it
has been shown that PD-1 ligation alters T-cell metabolic reprogramming by
inhibiting glycolysis and promoting lipolysis and fatty acid oxidation [97].
The study showed that PD-1 ligation promotes FAO of endogenous lipids by
increasing expression of CPT1A, and inducing lipolysis as indicated by
elevation of the lipase ATGL, the lipolysis marker glycerol and release of
fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO,
suggesting that CTLA-4 sustains the metabolic profile of non-activated cells.
While FAO is believed to promote T cell memory, it is unclear why PD-1
expressing T cells exhibit an exhausted phenotype. Is it that both memory and
exhausted T cells although rely on FAO, but have distinct metabolite signature
that would result in differences in longevity and ability to mount a recall
response? However, it must be noted that the PD-1 expression on T cells as
exclusive marker of exhaustion has already been called in question by elegant
studies where serial transfer of viral epitope specific PD-1 expressing T cells
were shown to still control the magnitude of infection [98]. Additionally,
blockade of PD-1 has been shown to decrease the efficacy of subsequent electron
transport chain [99], and this metabolic inhibition need to be considered when
using anti-PD-1 therapies in the clinic in order to augment long-term memory T cell
responses.

Given that
tumor cell themselves rely on the available glucose, it is likely that there is
a competition between the effector T cells infiltrating the tumor. Some recent
studies have elegantly shown that using the progressor or regressor tumors that
it is not only the more progressor tumor that is highly glucolytic [100], but
rapid depletion of available glucose by an aggressive tumor leads to a
dysfunctional and exhausted T cell (as identified by enhanced expression of
PD1). This study also showed that using antibodies against PD1 and CTLA4 – the
T cells are better able to control tumors since these check-point blockade
antibodies modulate the tumor metabolic commitment. Although, a question
remains that if CTLA4 and PD1 act through different mechanisms [101], then do
both pathways converge on the same metabolic pathway or a bifurcation point
exits in the metabolism to account for the differences in mechanism of action.

One of the
recent study has shown a new role for the glycolytic metabolite
phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca (2+)-NFAT
signaling and effector functions by repressing sarco/ER Ca (2+)-ATPase (SERCA)
activity. Tumor-specific CD4 and CD8 T cells could be metabolically
reprogrammed by increasing PEP production through overexpression of
phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions.
Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. Another
study has recently shown that α-ketoglutarate (αKG),the glutamine-derived
metabolite that enters into the mitochondrial citric acid cycle, acts as a
metabolic regulator of CD4+ T cell differentiation [102]. This study showed
that activation of naïve CD4+ T cells under conditions of glutamine deprivation
resulted in their differentiation into Foxp3+ regulatory T (Treg) cells, which
had suppressor function in vivo. Further, activation of glutamine-deprived
naïve CD4+ T cells in the presence of a cell-permeableαKG analog increased the
expression of the gene encoding the T-helper 1 (Th1) associated transcription
factor T-bet and resulted in their differentiation into Th1 cells, concomitant
with stimulation of mammalian target of rapamycin complex 1 (mTORC1) signaling.
Thus, this paper established that a decrease αKG, caused intracellular by the
amount of limited availability of extracellular glutamine, shifts the balance
between the generation of Th1 and Treg cells toward that of a Treg phenotype.
These above studies uncovers new metabolic checkpoints for T cell activity and
demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance
anti-tumor T cell responses, illuminating new forms of immunotherapy.

Targeting
glycolysis may not be always useful to control a T cell mediated disease state,
since T cells activated in vivo by alloantigens in graft-versus-host disease
(GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and
oxidation, with small increases of glucose uptake and aerobic glycolysis. Using
targeted metabolic 13C tracer fate association studies, to elucidate the
metabolic pathway(s) employed by alloreactive T cells in vivo it was found that
glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in
alloreactive T cells and that Gln carbon contributes to ribose biosynthesis.
This study further showed that pharmacological modulation of oxidative
phosphorylation rapidly reduces anaplerosis in alloreactive T cells and
improves GVHD [103]. Thus, T-cell metabolism is relevant to activated
lymphocytes in vivo, with implications for the discovery of new drugs for
immune disorders.

CONCLUSION

Various studies
with lymphoid and myeloid cells have highlighted the importance of
understanding metabolism to design better approach for intervention in disease
states. However, given the differences in metabolic fates between the lymphoid
subsets (as T vs. NK vs. B) or myeloid cells (DC vs. MDSC vs. macrophages), the
experimental strategies need to be carefully considered before reaching a
definitive conclusion of the metabolic commitment since the dynamic metabolic
states that depends upon activation state, activation signals, strength of
activation etc. could result in differences in metabolism or metabolite
accumulation that can lead to differences in function and viability of these
cellular subtypes.

ACKNOWLEDGEMENT

The work was
supported in part by funds from Department of Surgery and NIH grants
R21CA137725, R01CA138930, P01CA154778 to SM.